There is to date no systematic classification of megakaryocytes in terms of the development of platelet specific characteristics and functions. The proposed studies will define the cytokinetics of megakarocyte maturation in mouse femoral marrow and spleen using quantitative cellular measurements of DNA, RNA, cell size and platelet-associated proteins (PAP). Cell monodispersion conditions will be established for obtaining the optimal yield of intact, viable megakaryocytes that are representative of the tissue of origin as confirmed histologically. Megakaryocytes in suspension will be enriched selectively by centrifugal elutriation (CE) for multiparameter analysis by flow cytometry (FCM) and cell sorting using a B-D fluorescence-activated cell sorter (FACS IV) on the basis of: a) DNA content using vital DNA fluorochromes (Hoechst); b) RNA content using acridine orange; c) cell size by Coulter sensing and light scatter (FCM) and CE; d) the presence and content of PAP using fluorescently-labeled antibodies to platelet factor 4 and platelet-derived growth factor; and e) binding of fluorescently-tagged monoclonal anti-platelet antibody and a fluorescent probe for acetylcholinesterase activity as megakaryocyte-specific cytoplasmic markers. Analysis of metabolic activity will be carried out in terms of platelet-associated prostaglandin synthesis on total and sorted megakaryocyte sub-populations as a maturational index to be correlated with megakaryocyte DNA and RNA content, size and PAP-activity. The regulation of megakaryocyte maturation will be tested in the cytokinetic response of megakaryocytes in mice with altered platelet demands following immune-induced thrombocytopenia and irradiation as well as selective cytolysis of specific megakaryocyte sub-populations and analysis will be carried out within the context of a mathematical cell model of maturation.